Elucidating the roles of electrolytes and hydrogen bonding in the dewetting dynamics of the tear film.

This study explores the impact of electrostatic interactions and hydrogen bonding on tear film stability, a crucial factor for ocular surface health. While mucosal and meibomian layers have been extensively studied, the role of electrolytes in the aqueous phase remains unclear. Dry eye syndrome, characterized by insufficient tear quantity or quality, is associated with hyperosmolality, making electrolyte composition an important factor that might impact tear stability. Using a model buffer solution on a silica glass dome, we simulated physiologically relevant tear film conditions. Sodium chloride alone induced premature dewetting through salt crystal nucleation. In contrast, trace amounts of solutes with hydroxyl groups (sodium phosphate dibasic, potassium phosphate monobasic, and glucose) exhibited intriguing phenomena: quasi-stable films, solutal Marangoni-driven fluid influx increasing film thickness, and viscous fingering due to Saffman-Taylor instability. These observations are rationalized by the association of salt solutions with increased surface tension and the propensity of hydroxyl-group-containing solutes to engage in significant hydrogen bonding, altering local viscosity. This creates a viscosity contrast between the bulk buffer solution and the film region. Moreover, these solutes shield the glass dome, counteracting sodium chloride crystallization. These insights not only advance our understanding of tear film mechanics but also pave the way for predictive diagnostics in dry eye syndrome, offering a robust platform for personalized medical interventions based on individual tear film composition.

TMPyP binding evokes a complex, tunable nanomechanical response in DNA.

TMPyP is a porphyrin capable of DNA binding and used in photodynamic therapy and G-quadruplex stabilization. Despite its broad applications, TMPyP's effect on DNA nanomechanics is unknown. Here we investigated, by manipulating λ-phage DNA with optical tweezers combined with microfluidics in equilibrium and perturbation kinetic experiments, how TMPyP influences DNA nanomechanics across wide ranges of TMPyP concentration (5-5120 nM), mechanical force (0-100 pN), NaCl concentration (0.01-1 M) and pulling rate (0.2-20 µm/s). Complex responses were recorded, for the analysis of which we introduced a simple mathematical model. TMPyP binding, which is a highly dynamic process, leads to dsDNA lengthening and softening. dsDNA stability increased at low (<10 nM) TMPyP concentrations, then decreased progressively upon increasing TMPyP concentration. Overstretch cooperativity decreased, due most likely to mechanical roadblocks of ssDNA-bound TMPyP. TMPyP binding increased ssDNA's contour length. The addition of NaCl at high (1 M) concentration competed with the TMPyP-evoked nanomechanical changes. Because the largest amplitude of the changes is induced by the pharmacologically relevant TMPyP concentration range, this porphyrin derivative may be used to tune DNA's structure and properties, hence control the wide array of biomolecular DNA-dependent processes including replication, transcription, condensation and repair.

Evidence for a semisolid phase state of aerosols and droplets relevant to the airborne and surface survival of pathogens.

The phase state of respiratory aerosols and droplets has been linked to the humidity-dependent survival of pathogens such as SARS-CoV-2. To inform strategies to mitigate the spread of infectious disease, it is thus necessary to understand the humidity-dependent phase changes associated with the particles in which pathogens are suspended. Here, we study phase changes of levitated aerosols and droplets composed of model respiratory compounds (salt and protein) and growth media (organic-inorganic mixtures commonly used in studies of pathogen survival) with decreasing relative humidity (RH). Efflorescence was suppressed in many particle compositions and thus unlikely to fully account for the humidity-dependent survival of viruses. Rather, we identify organic-based, semisolid phase states that form under equilibrium conditions at intermediate RH (45 to 80%). A higher-protein content causes particles to exist in a semisolid state under a wider range of RH conditions. Diffusion and, thus, disinfection kinetics are expected to be inhibited in these semisolid states. These observations suggest that organic-based, semisolid states are an important consideration to account for the recovery of virus viability at low RH observed in previous studies. We propose a mechanism in which the semisolid phase shields pathogens from inactivation by hindering the diffusion of solutes. This suggests that the exogenous lifetime of pathogens will depend, in part, on the organic composition of the carrier respiratory particle and thus its origin in the respiratory tract. Furthermore, this work highlights the importance of accounting for spatial heterogeneities and time-dependent changes in the properties of aerosols and droplets undergoing evaporation in studies of pathogen viability.

Is Native Mass Spectrometry in Ammonium Acetate Really Native? Protein Stability Differences in Biochemically Relevant Salt Solutions.

Ammonium acetate is widely used in native mass spectrometry to provide adequate ionic strength without adducting to protein ions, but different ions can preferentially stabilize or destabilize the native form of proteins in solution. The stability of bovine serum albumin (BSA) was investigated in 50 mM solutions of a variety of salts using electrospray emitters with submicron tips to desalt protein ions. The charge-state distribution of BSA is narrow (+14 to +18) in ammonium acetate (AmmAc), whereas it is much broader (+13 to +42) in solutions containing sodium acetate (NaAc), ammonium chloride (AmmCl), potassium chloride (KCl), and sodium chloride (NaCl). The average charge state and percent of unfolded protein increase in these respective solutions, indicating greater extents of protein destabilization and conformational changes. In contrast, no high charge states of either bovine carbonic anhydrase II or IgG1 were formed in AmmAc or NaCl despite their similar melting temperatures to BSA, indicating that the presence of unfolded BSA in some of these solutions is not an artifact of the electrospray ionization process. The charge states formed from the nonvolatile salt solutions do not change significantly for up to 7 min of electrospray, but higher charging occurs after 10 min, consistent with solution acidification. Formation of unfolded BSA in NaAc but not in AmmAc indicates that the cation identity, not acidification, is responsible for structural differences in these two solutions. Temperature-dependent measurements show both increased charging and aggregation at lower temperatures in NaCl:Tris than in AmmAc, consistent with lower protein stability in the former solution. These results are consistent with the order of these ions in the Hofmeister series and indicate that data on protein stability in AmmAc may not be representative of solutions containing nonvolatile salts that are directly relevant to biology.

Flow Activation Energy of High-Concentration Monoclonal Antibody Solutions and Protein-Protein Interactions Influenced by NaCl and Sucrose.

The solution viscosity and protein-protein interactions (PPIs) as a function of temperature (4-40 °C) were measured at a series of protein concentrations for a monoclonal antibody (mAb) with different formulation conditions, which include NaCl and sucrose. The flow activation energy (Eη) was extracted from the temperature dependence of solution viscosity using the Arrhenius equation. PPIs were quantified via the protein diffusion interaction parameter (kD) measured by dynamic light scattering, together with the osmotic second virial coefficient and the structure factor obtained through small-angle X-ray scattering. Both viscosity and PPIs were found to vary with the formulation conditions. Adding NaCl introduces an attractive interaction but leads to a significant reduction in the viscosity. However, adding sucrose enhances an overall repulsive effect and leads to a slight decrease in viscosity. Thus, the averaged (attractive or repulsive) PPI information is not a good indicator of viscosity at high protein concentrations for the mAb studied here. Instead, a correlation based on the temperature dependence of viscosity (i.e., Eη) and the temperature sensitivity in PPIs was observed for this specific mAb. When kD is more sensitive to the temperature variation, it corresponds to a larger value of Eη and thus a higher viscosity in concentrated protein solutions. When kD is less sensitive to temperature change, it corresponds to a smaller value of Eη and thus a lower viscosity at high protein concentrations. Rather than the absolute value of PPIs at a given temperature, our results show that the temperature sensitivity of PPIs may be a more useful metric for predicting issues with high viscosity of concentrated solutions. In addition, we also demonstrate that caution is required in choosing a proper protein concentration range to extract kD. In some excipient conditions studied here, the appropriate protein concentration range needs to be less than 4 mg/mL, remarkably lower than the typical concentration range used in the literature.

Understanding the Roles of Excipients in Moisture Management in Solid Dosage Forms.

Excipients are ubiquitous in pharmaceutical products, and often, they can also play a critical role in maintaining product quality. For a product containing a moisture-sensitive drug, moisture can be deleterious to the product stability during storage. Therefore, using excipients that interact with moisture in situ can potentially alleviate product stability issues. In this study, the interactive behavior of starch with moisture was augmented by coprocessing maize starch with sodium chloride (NaCl) or magnesium nitrate hexahydrate [Mg(NO3)2·6H2O] at different concentrations (5 and 10%, w/w). The effect of the formulation on drug stability was assessed through the degradation of acetylsalicylic acid, which was used as the model drug. The results showed that coprocessing of the starch with either NaCl or Mg(NO3)2·6H2O impacted the number of water molecule binding sites on the starch and how the sorbed moisture was distributed. The coprocessed excipients also resulted in lower drug degradation and lesser changes in tablet tensile strength during post-compaction storage. However, corresponding tablet formulations containing physical mixtures of starch and salts did not yield promising outcomes. This study demonstrated the advantageous concomitant use of common excipients by coprocessing to synergistically mitigate the adverse effects of moisture and promote product stability when formulating a moisture-sensitive drug. In addition, the findings could help to improve the understanding of moisture-excipient interactions and allow for the judicious choice of excipients when designing formulations containing moisture-sensitive drugs.

Salt-Induced Hydrophobic C-Terminal Region of α-Synuclein Triggers Its Fibrillation under the Mimic Physiologic Condition.

α-Synuclein (αS) causes Parkinson's disease due to the structural alteration into amyloid fibrils that form after the interaction with synaptic membranes in neurons. To understand the alternation mechanism, the effect of salt (NaCl) on the interaction of αS with synaptic mimic membrane was characterized at the molecular level because salt triggered the amyloid fibril formation. The membrane-bound conformation (or the initial conformation before fibrillation) showed that NaCl decreased the number of helical structures and Tyr residues interacting with the membrane surface compared to when NaCl was absent, implying an increase in solvent-exposed regions. The N-terminal region of αS interacted with the membrane, forming the helical structures regardless of NaCl, while the C-terminal region formed a random structure with weak membrane interaction, but NaCl inhibited the interaction of its hydrophobic area, suggesting that salt promoted amyloid fibril formations by exposing the hydrophobic C-terminal region, which can intermolecularly interact with free αS.

Dialysis Monitoring of Ionic Strength and Denaturant Effects, and Their Reversibility, for Various Classes of Macromolecules.

Monitoring membrane-mediated dialysis in real time with static and dynamic light scattering revealed distinctive differences, including reversibility/irreversibility, in the effects of ionic strength (NaCl) and the denaturant guanidine-HCl (Gd) on a synthetic polyelectrolyte and several types of biomacromolecules: protein, polysaccharide, and polyampholyte. Dialysis cycles against aqueous NaCl and Gd, and reverse back to the original aqueous solution, were monitored. The behavior of Na-polystyrenesulfonate was reversible and yielded a detailed polymer physics description. The biomacromolecules additionally showed hydrogen-bonding/hydrophobic (HP) interactions. An interpretive model was developed that considers the interplay among polyelectrolyte, polyampholyte, and HP potential energies in determining the different associative, aggregative, and dissociative behaviors. NaCl isolated purely electrostatic effects, whereas Gd combined electrostatic and HP effects. Some macromolecules showed partially reversible behavior, and others were completely irreversible. The dialysis monitoring method should prove useful for investigating fundamental macromolecular and colloid properties and for drug formulation and stability optimization.

1H-NMR studies on the volume phase transition of DNA-modified pNipmam microgels.

DNA functionalized pNipmam microgels, which have recently been introduced, are examined at different concentrations of sodium chloride and in PBS solutions via temperature dependent 1H-NMR measurements and are compared to pure pNipmam microgels. We show that the DNA modification shifts the volume phase transition temperature towards lower temperatures and the addition of salt and PBS further supports this effect in both materials. Thermodynamic values, i.e. enthalpy, entropy and Gibbs free energy, are determined via a non-linear fit which can be applied directly to the measurement data without further linearization.

A Novel Size Exclusion Chromatography Method for the Analysis of Monoclonal Antibodies and Antibody-drug Conjugates by Using Sodium Iodide in the Mobile Phase.

PURPOSES: Size exclusion chromatography (SEC) is widely used to characterize molecular size variants of antibody drugs. However, SEC analysis is hindered by secondary interactions (or nonspecific interactions) between proteins and stationary phase packing, which result in poor column efficiency. Previous studies have reported that chaotropic salt can inhibit these interactions, but the corresponding applications of this aspect are relatively rare. Therefore, this study introduces a novel approach using sodium iodide (NaI) as a mobile-phase component in SEC and investigates the influence of the mobile-phase composition on secondary interactions. METHODS: SEC analysis was performed on one antibody-drug conjugate and four monoclonal antibodies (mAbs) using three different mobile-phase systems (i.e., sodium chloride/L-arginine hydrochloride/NaI mobile phases system) to compare the column efficiency. Subsequently, mAb-1 was used as a model to investigate the effects of these factors on secondary interactions by adjusting the ionic strength (salt concentration) and pH of the NaI mobile-phase system. RESULTS: NaI exhibits superior column efficiency performance in the SEC analysis of most products. The ionic strength will affect nonideal electrostatic and hydrophobic interaction. An appropriate ionic strength can inhibit electrostatic interactions, while an excessive ionic strength increases hydrophobic interactions. pH primarily influences electrostatic interactions. Determining the appropriate pH necessitates consideration of the isoelectric point of the protein and the pH tolerance of the column. CONCLUSIONS: In SEC analysis, using NaI as the salt component in the mobile phase reduces secondary interactions and improves column efficiency. This approach is advantageous for samples with intense secondary interactions and is a suitable alternative.

Analysis of the effect of cations on protein conformational stability using solid-state nanopores.

The conformation of proteins is closely related to their biological functions, and it is affected by many factors, including the type of cations in solution. However, it is difficult to detect the conformational changes of a protein in situ. As a single-molecule sensing technology, nanopores can convert molecular structural information into analyzable current signals within a reasonable time range. Herein, we detect and analyze the effects of two different types of monovalent cations (Na+ and Li+) on a model protein bovine serum albumin (BSA) conformation using SiNx nanopores with different diameters. The quantitative analysis results show that the excluded volume of BSA in LiCl salt solutions is larger than the value in NaCl solution, indicating that Li+ is more prone to unfolding the proteins and making them unstable. This study demonstrated that nanopores enable the in situ detection of the structure of proteins at the single-molecule level and provide a new approach for the quantitative analysis of proteins.

Nanoplastics Detected in Commercial Sea Salt.

People of all ages consume salt every day, but is it really just salt? Plastic nanoparticles [nanoplastics (NPs)] pose an increasing environmental threat and have begun to contaminate everyday salt in consumer goods. Herein, we developed a combined surface enhanced Raman scattering (SERS) and stimulated Raman scattering (SRS) approach that can realize the filtration, enrichment, and detection of NPs in commercial salt. The Au-loaded (50 nm) anodic alumina oxide substrate was used as the SERS substrate to explore the potential types of NP contaminants in salts. SRS was used to conduct imaging and quantify the presence of the NPs. SRS detection was successfully established through standard plastics, and NPs were identified through the match of the hydrocarbon group of the nanoparticles. Simultaneously, the NPs were quantified based on the high spatial resolution and rapid imaging of the SRS imaging platform. NPs in sea salts produced in Asia, Australasia, Europe, and the Atlantic were studied. We estimate that, depending on the location, an average person could be ingesting as many as 6 million NPs per year through the consumption of sea salt alone. The potential health hazards associated with NP ingestion should not be underestimated.

Imaging Dissolution Dynamics of Individual NaCl Nanoparticles during Deliquescence with In Situ Transmission Electron Microscopy.

Water vapor condensation on hygroscopic aerosol particles plays an important role in cloud formation, climate change, secondary aerosol formation, and aerosol aging. Conventional understanding considers deliquescence of nanosized hygroscopic aerosol particles a nearly instantaneous solid to liquid phase transition. However, the nanoscale dynamics of water condensation and aerosol particle dissolution prior to and during deliquescence remain obscure due to a lack of high spatial and temporal resolution single particle measurements. Here we use real time in situ transmission electron microscopy (TEM) imaging of individual sodium chloride (NaCl) nanoparticles to demonstrate that water adsorption and aerosol particle dissolution prior to and during deliquescence is a multistep dynamic process. Water condensation and aerosol particle dissolution was investigated for lab generated NaCl aerosols and found to occur in three distinct stages as a function of increasing relative humidity (RH). First, a < 100 nm water layer adsorbed on the NaCl cubes and caused sharp corners to dissolve and truncate. The water layer grew to several hundred nanometers with increasing RH and was rapidly saturated with solute, as evidenced by halting of particle dissolution. Adjacent cube corners displayed second-scale curvature fluctuations with no net particle dissolution or water layer thickness change. We propose that droplet solute concentration fluctuations drove NaCl transport from regions of high local curvature to regions of low curvature. Finally, we observed coexistence of a liquid water droplet and aerosol particle immediately prior to deliquescence. Particles dissolved discretely along single crystallographic directions, separated by few second lag times with no dissolution. This work demonstrates that deliquescence of simple pure salt particles with sizes in the range of 100 nm to several microns is not an instantaneous phase transition and instead involves a range of complex dissolution and water condensation dynamics.

Developing Salt-Rejecting Evaporators for Solar Desalination: A Critical Review.

Solar desalination, a green, low-cost, and sustainable technology, offers a promising way to get clean water from seawater without relying on electricity and complex infrastructures. However, the main challenge faced in solar desalination is salt accumulation, either on the surface of or inside the solar evaporator, which can impair solar-to-vapor efficiency and even lead to the failure of the evaporator itself. While many ideas have been tried to address this ″salt accumulation″, scientists have not had a clear system for understanding what works best for the enhancement of salt-rejecting ability. Therein, for the first time, we classified the state-of-the-art salt-rejecting designs into isolation strategy (isolating the solar evaporator from brine), dilution strategy (diluting the concentrated brine), and crystallization strategy (regulating the crystallization site into a tiny area). Through the specific equations presented, we have identified key parameters for each strategy and highlighted the corresponding improvements in the solar desalination performance. This Review provides a semiquantitative perspective on salt-rejecting designs and critical parameters for enhancing the salt-rejecting ability of dilution-based, isolation-based, and crystallization-based solar evaporators. Ultimately, this knowledge can help us create reliable solar desalination solutions to provide clean water from even the saltiest sources.

Salt Supersaturation as an Accelerator of Influenza A Virus Inactivation in 1 µL Droplets.

Influenza A virus (IAV) spreads through exhaled aerosol particles and larger droplets. Estimating the stability of IAV is challenging and depends on factors such as the respiratory matrix and drying kinetics. Here, we combine kinetic experiments on millimeter-sized saline droplets with a biophysical aerosol model to quantify the impact of NaCl on IAV stability. We show that IAV inactivation is determined by NaCl concentration, which increases during water evaporation and then decreases again when efflorescence occurs. When drying in air with relative humidity RH = 30%, inactivation follows an inverted sigmoidal curve, with inactivation occurring most rapidly when the NaCl concentration exceeds 20 mol/(kg H2O) immediately prior to efflorescence. Efflorescence reduces the NaCl molality to saturated conditions, resulting in a significantly reduced inactivation rate. We demonstrate that the inactivation rate k depends exponentially on NaCl molality, and after the solution reaches equilibrium, the inactivation proceeds at a first-order rate. Introducing sucrose, an organic cosolute, attenuates IAV inactivation via two mechanisms: first by decreasing the NaCl molality during the drying phase and second by a protective effect against the NaCl-induced inactivation. For both pure saline and sucrose-containing droplets, our biophysical model ResAM accurately simulates the inactivation when NaCl molality is used as the only inactivating factor. This study highlights the role of NaCl molality in IAV inactivation and provides a mechanistic basis for the observed inactivation rates.

Perovskite nickelates as electric-field sensors in salt water.

Designing materials to function in harsh environments, such as conductive aqueous media, is a problem of broad interest to a range of technologies, including energy, ocean monitoring and biological applications. The main challenge is to retain the stability and morphology of the material as it interacts dynamically with the surrounding environment. Materials that respond to mild stimuli through collective phase transitions and amplify signals could open up new avenues for sensing. Here we present the discovery of an electric-field-driven, water-mediated reversible phase change in a perovskite-structured nickelate, SmNiO3. This prototypical strongly correlated quantum material is stable in salt water, does not corrode, and allows exchange of protons with the surrounding water at ambient temperature, with the concurrent modification in electrical resistance and optical properties being capable of multi-modal readout. Besides operating both as thermistors and pH sensors, devices made of this material can detect sub-volt electric potentials in salt water. We postulate that such devices could be used in oceanic environments for monitoring electrical signals from various maritime vessels and sea creatures.

Synthesis, characterization and application of chitosan functionalized and functional graphene oxide membranes for desalination of water by pervaporation.

The use of graphene sheets in water treatment is increasing due to its adsorption capacity, reactivity, catalytic action and surface area. The challenges linked to wastewater treatment are vast due to the constant influx of various pollutants. Can the challenges of water desalination and purification be encountered by graphene-based composites and membranes?.The current work describes the synthesis of graphene oxide (GO) using modified Hummers' method. GO was functionalized with chitosan and used as adsorbents. On the other hand, it was reported that the surface of thin-film-composite (TFC) polyamide membranes was modified in order to desalinate highly saline water using pervaporation. The findings showed that GO synthesized by modified Hummers' method has a greater capacity for the adsorption of sodium ion and have better regeneration performance. Functionalization with chitosan increased adsorption capacity from 680.2 to 740.5 mg/g at the initial concentration of 45,000 mg/l of Na+ ions. On the other hand, modification in membrane comprises the chlorine treatment of surface of polyamide membrane. Layer-by-layer (LbL) deposition of positively charged polyethyleneimine (PEI) and negatively charged graphene oxide (GO) was followed. The PEI/GO LbL membrane's pure water flux was twice as high as compare to the original membrane. The synthesized membrane was tested against the aqueous solutions containing Na2SO4, MgSO4, NaCl and MgCl2 salts for their desalination. At different concentrations, a water flux of 8.9 kg/m2h with a huge salt rejection (>99.9%) was attained for every tested salt. It was observed that CS functionalized GO and GO membrane showed higher adsorption capacity and improved regeneration performance can be measured as an operational and active adsorbent for sea water desalination.

Sensitive osteoarthritis sensing by salt-induced aggregation and dispersion of gold nanoparticles.

Osteoarthritis occurs in any joints, and identification in its earlier stages helps to treat the disease and increase the recovery rate. The radiography method and imaging techniques are traditionally used to identify osteoarthritis. But these methods are expensive, and with the complicated steps. Researchers are working toward developing a highly sensitive biosensor in identifying the osteoarthritis biomarker. This research was focused on developing a C-terminal telopeptide of type II collagen (CTX-II) colorimetric sensor with gold nanoparticle (AuNP) for diagnosing osteoarthritis. Anti-CTX-II was conjugated with AuNP and then added with CTX-II and sodium chloride for the color change. In the presence of CTX-II, antibody releases from AuNP then binds with CTX-II, and the color of AuNP changed to purple. Without the CTX-II, AuNP remains its red color (dispersed). This easier colorimetric assay detected the CTX-II as low as 2 ng/mL on linear regression [y = 0.0131x - 0.0051; R2 = 0.9205]. Furthermore, control performances with the relevant proteins osteopontin, IL-6, and nonimmune antibody failed to change the color confirming the specific identification of CTX-II.

Probing the structural evolution on the surface of cardiolipin vesicles with an amphiphilic second harmonic generation and fluorescence probe.

Investigating the influence of the ambient chemical environment on molecular behaviors in liposomes is crucial for understanding and manipulating cellular vitality as well as the capabilities of lipid drug carriers in various environments. Here, we designed and synthesized a second harmonic generation (SHG) and fluorescence probe molecule called Pyr-Py+-N+ (PPN), which possesses membrane-targeting capability. We employed PPN to investigate the response of lipid vesicles composed of cardiolipin to the presence of exogenous salt. The kinetic behaviors, including the adsorption and embedding of PPN on the surface of small unilamellar vesicles (SUVs) composed of cardiolipin, were analyzed. The response of the SUVs to the addition of NaCl was also monitored. A rapid decrease in vesicle size can be evidenced through the rapid drop in SHG emission originating from PPN located on the vesicle surface.

Extended physicochemical stability of cetuximab in opened vials and infusion bags when stored at 4°C and 25°C.

INTRODUCTION: This study aimed to determine the stability of cetuximab: (1) under "in-use" conditions after dilution to 1 mg/mL in 0.9% sodium chloride in polyolefin bags and (2) as an undiluted solution (5 mg/mL) repackaged in polypropylene bags or kept in the vial after opening. METHODS: Ready-to-use 500 mg/100 mL vials of cetuximab solution were diluted to 1 mg/mL in 100 mL bags of 0.9% sodium chloride or repackaged as a 5 mg/mL solution into empty 100 mL bags. Bags and vials were stored at 4°C for 90 days and 25°C for 3 days. A syringe sample of 7 mL was taken from each bag for the initial determinations. The sampled bags were weighed to determine their initial weight and placed under the planned storage conditions. The physicochemical stability of cetuximab was estimated using validated methods. RESULTS: No changes in turbidity, no protein loss, and no changes in cetuximab tertiary structure were observed after 30 days of storage or when subjected to a temperature excursion of 3 days at 25°C and when stored at 4°C for up to 90 days, regardless of the concentrations and batches. The colligative parameters did not change under any of the tested conditions. No evidence of microbial growth was found in bags after 90 days of storage at 4°C. CONCLUSION: These results support the extended in-use shelf-life of cetuximab vials and bags, which can be cost-effective for healthcare providers.